US20150266927A1 - High efficiency method for purifying human papillomavirus virus-like particles - Google Patents

High efficiency method for purifying human papillomavirus virus-like particles Download PDF

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US20150266927A1
US20150266927A1 US14/418,004 US201314418004A US2015266927A1 US 20150266927 A1 US20150266927 A1 US 20150266927A1 US 201314418004 A US201314418004 A US 201314418004A US 2015266927 A1 US2015266927 A1 US 2015266927A1
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US9994618B2 (en
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Hong-Jin Kim
Hyoung Jin Kim
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Posvax Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20051—Methods of production or purification of viral material

Definitions

  • the present invention relates to a method of purifying virus-like particles (VLPs) of human papillomavirus (HPV) having excellent structural and immunological characteristics with high efficiency.
  • VLPs virus-like particles
  • HPV human papillomavirus
  • HPV is a pathogen causing approximately 100% of cervical cancer cases [1]. It is reported that, annually, 500,000 women are diagnosed with cervical cancer around the world, and 250,000 women die from cervical cancer [2]. Types 16, 18, 45, 31, 33, 52, 58, 35, and 59 are known as high risk types of HPV that cause cervical cancer, whereas types 6 and 11 are known as low risk types of HPV [3,4]. In particular, HPV16 and HPV18 are known to cause 70% of all cervical cancer cases, and thus are recognized as the most important types in prevention of cervical cancer [5]. Types of HPVs causing cervical cancer vary by region [5].
  • HPV16, HPV18, HPV31 and HPV45 infections are main causes of cervical cancer
  • HPV16, HPV18, HPV58 and HPV33 infections are main causes of cervical cancer [5].
  • a capsid of HPV is composed of L1 protein as a major antigen and L2 protein as a minor antigen [6].
  • the L1 protein has been used as an antigen for prophylactic vaccine of cervical cancer and an antigen for diagnosis because it has a property of self-assembly that forming virus-like particles (VLPs) [6, 27].
  • Recombinant L1 protein was produced in Escherichia coli, Saccharomyces cerevisiae, Pichia Pastoris, Lactobacillus casei , or Spodoptera frugiperda (Sf) cells, or plant cells as expression cells [7-12, 28].
  • GardasilTM Today, commercially available cervical cancer vaccines are GardasilTM (Merck) and CervarixTM (GlaxosmithKline, GSK).
  • GardasilTM includes L1 VLPs with respect to HPV16, HPV18, HPV6 and HPV11 as antigens
  • CervarixTM includes L1 VLPs with respect to HPV16 and HPV18 as antigens [13].
  • GardasilTM uses Saccharomyces cerevisiae as an antigen-expressing cell
  • CervarixTM uses Spodoptera frugiperda (Sf) cells, which are insect cells, as antigen-expressing cells [13,14]. Both of the two vaccines are injected by intramuscular injection, and have high costs of $120 for one dose and $360 for three doses [15].
  • the vaccines Due to the high injection costs of the commercially available cervical cancer vaccines, the vaccines have many limits to be widely used in a developing country, which is a region in which cervical cancer mainly occurs [16]. Accordingly, development of a low-cost and highly-effective cervical cancer vaccine still remains an important issue.
  • a cost for downstream processing is as high as 80% of the total production cost [17].
  • a method of sequentially increasing purity of a target antigen through several steps of purification in the downstream processing is used [18].
  • sucrose cushion using ultracentrifugation or size-exclusion chromatography has been mainly used.
  • Hofmann et al. used a sucrose cushion using ultracentrifugation, anion exchange chromatography, ammonium sulfate precipitation and size-exclusion chromatography [19].
  • the inventors made an effort to develop a new method of purifying HPV L1 proteins produced from a host cell expressing a HPV L1 protein with high purity and high efficiency.
  • HPV L1 proteins were purified by chromatography including treating a homogenate of the host cell expressing the HPV L1 protein with a reducing agent, or treating a homogenate with a reducing agent and performing heating and chilling, a purity of the HPV L1 proteins may be remarkably enhanced, and structural and immunological characteristics of VLPs assembled from the L1 proteins may also be remarkably enhanced was confirmed by an experiment, and therefore the present invention was completed.
  • the present invention is directed to providing a method of purifying HPV L1protein with high purity and high efficiency.
  • the present invention provides a method of purifying HPV L1 proteins, which includes: (a) culturing transformed host cells expressing an HPV L1 protein, harvesting the cultured host cells, and disrupting the cells; (b) adding a reducing agent to a homogenate of the host cells; and (c) purifying the HPV L1 proteins by performing chromatography to the homogenate of the host cells to which the reducing agent is added.
  • a method of purifying HPV L1 proteins includes (i) culturing transformed host cells expressing an HPV L1 protein, harvesting the cultured host cells and disrupting the cells; (ii) adding a reducing agent to the homogenate of the host cells; (iii) heating and chilling the homogenate of the host cells to which the reducing agent is added; and (iv) purifying HPV L1 proteins by performing chromatography to the heated and chilled homogenate of the host cells.
  • HPV L1 protein refers to a major protein constituting a HPV capsid, which is expressed from an L1 gene of HPV.
  • the L1 proteins self-assembles into a VLPs alone or does in combination with a minor proteins, L2 proteins, to construct the capsids.
  • Papillomavirus is an icosahedral DNA genome virus, which has a maximum eight early genes (E) and two late genes (L), and a size of 50 to 60 nm, and no envelope.
  • E early genes
  • L late genes
  • the E gene is a gene involved in virus replication and transformation.
  • the L1 and L2 genes encode a virus capsid protein.
  • the L1 protein is a major capsid protein, and has a molecular weight of 55 to 60 kDa.
  • the L2 protein is a minor capid protein having an estimated molecular weight of 55 to 60 kDa and an apparent molecular weight of 75 to 100 kDa, measured by PAGE.
  • a type of HPV from which the L1 protein is derived may be selected from, but is not particularly limited to, the group consisting of HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 30, HPV type 31, HPV type 33, HPV type 35, HPV type 39, HPV type 41, HPV type 42, HPV type 43, HPV type 44, HPV type 45, HPV type 51, HPV type 52, HPV type 54, HPV type 55, HPV type 56, HPV type 58, HPV type 68 and HPV type 70, and more preferably, the L1 protein of the present invention may be derived from HPV selected from the group consisting of HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 45 and HPV type 58, and most preferably, derived from HPV type 16, HPV type 18, or HPV type 58.
  • cells used as host cells are bacteria, yeast cells, insect cells, plant cells or animal cells.
  • the yeast cell is Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces sp., Schizosaccharomyces pombe, Pichia Pastoris , or Hansenula polymorpha.
  • the transformed host cells expressing an HPV L1 protein are host cells transformed by an expression vector successfully expressing an HPV L1 protein.
  • the expression vector may include a transcription or translation regulatory factor known in the art, or a marker gene.
  • the transformed host cell expressing the HPV L1 protein of the present invention may be easily manufactured using a known method in the art.
  • reducing agent refers to an element or compound donating electrons to other species in a reduction-oxidation reaction, and preferably, a compound used to reduce a disulfide bond in a peptide or protein or stabilize a free sulfhydryl group.
  • the reducing agent is selected from, for example, the group consisting of ⁇ -mercaptoethanol, dithiothreitol (DTT), 2-mercaptoethylamine-HCl, tris(2-carboxyethyl)phosphine (TCEP) and cysteine-HCl,and preferably, ⁇ -mercaptoethanol or DTT.
  • the final concentration of the reducing agent in the cell homogenate may be 0.1 wt % or more, preferably, 0.1 to 20 wt %, more preferably 0.1 to 10 wt %, further more preferably 1 to 8 wt %, still more preferably 3 to 7 wt %, and most preferably 4 to 6 wt %.
  • a time for reacting the reducing agent with the cell homogenate is not limited to a specific range of time, and one of ordinary skill in the art may select a reaction time suitable for the method of the present invention.
  • the reducing agent is added to the cell homogenate, and heating and chilling is performed.
  • the cell homogenate to which the reducing agent is added is heated and chilled, efficiency of removing impurities from the homogenate of the transformed host cells is remarkably increased, and a VLP assembled from the L1 proteins has a preferable structure and immunological characteristic.
  • a heating temperature of the cell homogenate is higher than room temperature, preferably, more than 25° C., more preferably, more than 25 to less than 80° C., further more preferably, 30 to 65° C., still more preferably 35 to 65° C., and most preferably 35 to 60° C., and the optimal heating condition is 35 to 55° C.
  • the heating time of the cell homogenate may be changed according to the heating temperature, and may be suitably selected in the range of 10 minutes to 72 hours, but the present invention is not limited to that range.
  • the heating time is preferably 30 minutes to 72 hours, more preferably, 30 minutes to 48 hours, further more preferably, 30 minutes to 24 hours, and most preferably, 30 minutes to 12 hours.
  • the chilling temperature is a temperature at which a sample of the cell homogenate is not frozen.
  • a chilling temperature is 0 to 10° C., preferably, more than 0° C. to less than 8° C., more preferably, more than 0° C. to less than 7° C., further more preferably, more than 0° C. to less than 6° C., and most preferably, more than 0° C. to less than 5° C.
  • Chilling time may be used by selecting a chilling time suitable for the method of the present invention.
  • the homogenate of the host cells to which the reducing agent is added may not be heated and chilled, and maintained at room temperature.
  • room temperature used herein is a non-heated or non-chilled atmospheric temperature, which is 15 to 25° C.
  • HPV L1 proteins are purified by performing chromatography-based purification on the homogenate of the host cells to which the reducing agent is added, or by performing chromatography-based purification on the homogenate of the host cells after adding a reducing agent to the homogenate and heating and chilling the homogenate.
  • chromatography is known in the art, and may be, but is not limited to, for example, ion exchange chromatography such as cation exchange chromatography or anion exchange chromatography, size-exclusion chromatography (SEC), hydrophobic interaction chromatography, or affinity chromatography.
  • ion exchange chromatography which is most suitable for separation of a protein or peptide is preferably used because a substance to be separated and purified is a protein.
  • a type of the cation exchange chromatography, heparin resin chromatography or cation exchange chromatography was used to successfully separate and purify HPV L1 proteins.
  • the present invention provides a method of purifying HPV L1 proteins with high purity and high efficiency.
  • the purification method of the present invention is characterized by purifying L1 proteins by chromatography after a reducing agent is added to a cell homogenate of HPV L1 protein-expressing transformed host cells, or after performing heating and chilling after treatment with reducing agent to the cell homogenate.
  • a purification purity of HPV L1 protein may be remarkably enhanced.
  • VLPs of the HPV L1 proteins purified by the purification method of the present invention form a high quality structure, and have very excellent immunogenicity.
  • the present invention relates to a method of purifying HPV L1 proteins with high purity and high efficiency. According to the purification method of the present invention, a purification purity of the HPV L1 proteins may be remarkably enhanced, and since VLPs of the purified HPV L1 protein form a more similar structure to that of an original HPV virion, the VLPs have very excellent immunogenicity.
  • FIG. 1 shows a method described in a prior art document to purify HPV VLPs (hereinafter, referred to as a “T-1” method) and a method of the present invention (hereinafter, referred to as a “T-5” method).
  • T-1 prior art document to purify HPV VLPs
  • T-5 a method of the present invention
  • L1 proteins in the Saccharomyces cerevisiae homogenate were recovered by ammonium sulfate precipitation. Impurities in the recovered fraction were removed by removal of precipitated contaminants.
  • the L1 proteins in the homogenate processed through the above process were purified by heparin chromatography.
  • FIG. 2 shows a purification result obtained by the T-5 method including dialysis of a homogenate of a host cells expressing L1 proteins, treating the dialyzed result with a reducing agent and performing heating and chilling, performing first heparin chromatography, and the chromatography result was confirmed by SDS-PAGE. It appeared that high purity of HPV L1 proteins were recovered after the first heparin chromatography.
  • LS indicates loading sample that was loaded onto a heparin resin
  • FT indicates a flow-through, which is a fraction flowing through without binding to the heparin resin.
  • W indicates a wash, which is a fraction flowing through while the heparin resin is washed
  • E indicates an elution, which is a fraction in which HPV L1 protein is eluted with a buffer solution including 1M NaCl.
  • FIG. 3 shows a purification result obtained by the T-5 method, including directly treating a homogenate of HPV L1 protein-expressing host cells with a reducing agent without dialysis of the cell homogenate, performing heating and chilling, and performing first heparin chromatography.
  • the chromatography result was confirmed by SDS-PAGE. Similar to the result of FIG. 2 , high purity HPV L1 proteins were also obtained after the first heparin chromatography. LS, FT, W and E were the same as described in FIG. 2 .
  • FIG. 4 a shows a purification result obtained by the T-5 method, including treating a homogenate of HPV L1 protein expressing host cells with a reducing agent, performing heating and chilling, performing first heparin chromatography and performing second heparin chromatography.
  • the result was confirmed by SDS-PAGE.
  • LS and FT are the same as described in FIG. 2 .
  • Numerals from 3 to 18 shown in an upper part of the SDS-PAGE image indicate respective numbers of fractions obtained in a linear gradient elution using NaCl.
  • FIG. 4 b shows a profile of the second heparin chromatography.
  • FT flow-through
  • FIG. 5 shows an SDS-PAGE result for heparin chromatography according to the T-1 method.
  • Treatment of a sample before the heparin chromatography was performed as shown in FIG. 1 .
  • LS indicates a loading sample.
  • Proteins bound with a heparin resin were eluted by a method of linear gradient of increasing NaCl.
  • the linear gradient elution was performed to have a content of NaCl from 0.325 M to 2 M.
  • numerals of the upper part indicate numbers of elution fractions. It is shown that, in fractions 11 to 14, an L1 protein was eluted with high purity.
  • FIG. 6 a shows a comparison result between purities of VLPs purified by the conventional method (T-1 method) and the T-5 method.
  • the “T-1 HPV16 VLP” is a product purified by a conventional known method [Kim et al. (2010) Protein Expr Purif 70: 68-74; Kim et al. (2009) Arch Pharm Res 32: 1759-1766], and the “T-5 HPV16 VLP” is a product purified by the method of the present invention.
  • SDS-PAGE analysis of the HPV L1 proteins quantification of the protein was performed, and proteins were loaded at 500 ng, 250 ng, 125 ng and 62 ng per well. Independently, an experiment was performed twice, and respective experiments were represented as Panel A and Panel B. According to the results obtained by the two experiments, it was seen that an intensity of an L1 protein band of the T-5 HPV 16 VLPs was much higher than that of the T-1 HPV 16 VLPs.
  • FIG. 6 b are a graph showing intensities of L1 protein bands of T-1 HPV 16 VLP and T-5 HPV 16 VLP detected in the two experiments shown in FIG. 6 a .
  • the results were presented as mean ⁇ standard deviation, and the intensity of the L1 protein band of T-5 HPV 16 VLP loaded at 500 ng per well was set at 100%. This result shows that the purity of VLPs purified by the T-5 method is far superior to that of the VLPs purified by the T-1 method.
  • FIG. 7 shows a SDS-PAGE result when T-1 HPV 16 VLPs were loaded to have the same intensity of an L1 protein band as that of T-5 HPV 16 VLPs.
  • FIG. 8 shows electron microscopy results for T-1 HPV16 VLPs and T-5 HPV 16 VLPs. It was confirmed that the T-1 HPV 16 VLPs had a size of 20 to 50 nm, and the T-5 HPV16 VLPs had a size of 40 to 65 nm. This denotes that the size of the T-5 HPV 16 VLP is closer to an original size of HPV (50 to 60 nm) [29, 30].
  • FIGS. 9 and 10 show dynamic light scattering (DLS) results.
  • the DLS measures a size of VLP in a solution. Size distribution of VLPs in the solution was analyzed using DLS-700 ( FIG. 9 ) and ELS-Z2 ( FIG. 10 ) systems. As shown in the results of FIGS. 9 and 10 , it appeared that size distributions of the two types of VLPs are different from each other. The result shows that physical properties of the T-5 HPV 16 VLPs are different from those of the T-1 HPV16 VLPs.
  • FIG. 11 shows reactivity of monoclonal antibodies with respect to T-1 HPV 16 L1 VLP and T-5 HPV16 L1 VLP.
  • H16.V5 and H16.E70 previously known monoclonal antibodies, H16.V5 and H16.E70, were used.
  • An increase in reactivity of VLPs with respect to the two antibodies is known to have a close relationship with an increase in immunogenicity [26, 32-34]. It was confirmed that the reactivity of T-5 HPV 16 VLPs with respect to the H16.V5 and H16.E70 antibodies is far higher than those of T-1 HPV 16 VLPs.
  • FIG. 12 shows results obtained by analyzing immunogenicities of T-1 HPV16 L1 VLPs and T-5 HPV16 L1 VLPs.
  • 1 ng of VLPs was subcutaneously injected into a mouse with 200 ⁇ g of aluminum hydroxide. The immunization was performed four times at two week intervals. 10 days after the third and fourth immunizations, anti-HPV 16 L1 IgG antibody titers were detected from a serum.
  • anti-HPV 16 L1 IgG antibody titration it was confirmed that the level of anti-HPV16 L1 IgG induced by T-5 HPV16 L1 VLPs is 10 times higher than that of T-1 HPV16 L1 VLPs.
  • FIG. 13 shows anti-HPV16 neutralizing antibody activity of serum collected after fourth immunization in the immunization performed in FIG. 12 .
  • a neutralizing activity of a mouse serum immunized with T-5 HPV16 L1 VLPs was 78%, whereas a neutralizing activity of a mouse serum immunized with T-1 HPV16 L1 VLPs was 33%.
  • FIG. 14 shows comparison of purities of an HPV L1 protein purified when a homogenate was not treated (non-treatment), a homogenate was treated with a reducing agent ( ⁇ -ME), and when a homogenate was heated and chilled after treatment with a reducing agent ( ⁇ -ME+ heating and chilling).
  • ⁇ -ME reducing agent
  • ⁇ -ME+ heating and chilling a homogenate was heated and chilled after treatment with a reducing agent
  • FIG. 15 shows comparison of purities of an HPV L1 protein purified when a homogenate was not treated (non-treatment), when a homogenate was treated with a reducing agent ( ⁇ -ME), and when a homogenate was heated and chilled after treatment with a reducing agent (T-5, ⁇ -ME+ heating and chilling).
  • a sample going through each condition was eluted by first heparin chromatography, the same amount (1, 0.5 or 25 ⁇ g) each of the protein was taken from the elution fraction and loaded in a gel to perform SDS-PAGE and Western blotting.
  • FIG. 15 it was confirmed that a purity of the HPV L1 protein when a homogenate was treated with a reducing agent, heated and chilled (T-5, ⁇ -ME+ heating and chilling) is far higher than those in the other conditions.
  • FIG. 16 shows comparison of purities of an HPV L1 protein purified when a homogenate was not treated (non-treatment), when a homogenate was not treated with a reducing agent, but was only heated and chilled (heating and chilling), and when a homogenate was treated with a reducing agent, heated and chilled (T-5, ⁇ -ME+ heating and chilling).
  • a sample going through each condition was eluted by first heparin chromatography, the same volume (5, 2.5 or 1.2 ⁇ l) each of the protein, for each condition, was taken from the elution fraction and loaded in a gel to perform SDS-PAGE and Western blotting. According to the result of FIG. 16 , it was confirmed that a purity of the HPV L1 protein when a homogenate was treated with a reducing agent, heated and chilled (T-5, ⁇ -ME+ heating and chilling) is far higher than those in the other conditions.
  • FIG. 17 shows a purification method based on size-exclusion chromatography (SEC), a purification method based on ammonium sulfate precipitation, and a purification method performed by heating and chilling after treatment with a reducing agent (T-5, ⁇ -ME+ heating & chilling).
  • SEC size-exclusion chromatography
  • T-5 ⁇ -ME+ heating & chilling
  • T-5 ammonium sulfate precipitation
  • FIG. 18 shows a difference between ammonium sulfate precipitation, a reducing agent-treatment method ( ⁇ -ME), and a purification method performed by treatment with a reducing agent and heating/chilling (T-5, ⁇ -ME+ heating & chilling). Purity of elution fraction of first chromatography in each purification condition was analyzed by SDS-PAGE and Western blotting. As a result, it was confirmed that an L1 protein recovered by the method including treatment with ⁇ -ME and heating/chilling has the highest purity.
  • FIG. 19 shows first heparin chromatography results for the purification method according to ammonium sulfate precipitation and the purification method performed by heating and chilling after treatment with a reducing agent (T-5, ⁇ -ME+ heating & chilling).
  • a reducing agent T-5, ⁇ -ME+ heating & chilling
  • FIG. 20 shows SDS-PAGE and Western blotting results for elution fractions obtained by SEC in the purification method according to the SEC.
  • fractions 3 to 9 L1 proteins were eluted with high purity, and thus fractions in this section were collected to compare.
  • FIG. 21 shows SDS-PAGE and Western blotting results for L1 protein elution fractions obtained in first chromatography in the purification method according to SEC (SEC), the purification method according to ammonium sulfate precipitation, and the purification method performed by treatment with a reducing agent and heating/chilling (T-5, ⁇ -ME+ heating & chilling).
  • SEC SEC
  • ammonium sulfate precipitation the purification method performed by treatment with a reducing agent and heating/chilling
  • T-5 ⁇ -ME+ heating & chilling
  • the cell homogenate was subjected to the step removal of precipitated contaminants after the ammonium sulfate precipitation, and then subjected into heparin chromatography (first chromatography).
  • first chromatography For the method performed by the treatment with a reducing agent and heating/chilling, the homogenate was treated with a reducing agent, heated and chilled, and went through the heparin chromatography (first chromatography).
  • first chromatography For analysis of an elution fraction obtained by the first chromatography, a fraction for each condition was loaded at the same volume (0.35, 0.17 or 0.08 ⁇ l). According to the analysis result, it appeared that the L1 proteins obtained by the method including the treatment with a reducing agent and heating/chilling have the highest purity.
  • FIG. 22 shows a result obtained by further purifying the L1 protein from elution fraction obtained by the first chromatography of FIG. 21 by second chromatography.
  • the difference between L1 protein elution fractions after the second chromatography was analyzed by SDS-PAGE and Western blotting.
  • an elution fraction for each purification condition was loaded at the same volume (2, 1 or 0.5 ⁇ l). According to the analysis result, it was seen that the purification method including treatment with a reducing agent and heating/chilling was the best with respect to the recovery rate of L1 protein.
  • FIG. 23 shows reactivity of a monoclonal antibody (H16.E70) towards the L1 proteins finally purified in FIG. 22 , which was analyzed by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 24 shows analyses of immunogenicities of a purification method according to SEC, a purification method according to ammonium sulfate precipitation, and a purification method performed by treatment with a reducing agent and heating/chilling.
  • An amount of L1 protein before a mouse was immunized with HPV16 L1 VLP was adjusted to the same as detected in FIG. 23A .
  • 1 ng of HPV16 L1 VLPs was mixed with 200 ⁇ g of aluminum hydroxide, and then injected subcutaneously into the mouse. The mouse immunization was performed four times at two week intervals.
  • FIG. 25 shows an effect as a function of heating temperature in the process of heating/chilling after treatment with a reducing agent in T-5 method.
  • a cell homogenate was heated at each heating temperature for 15 minutes and chilled, and then the precipitated contaminants was removed by centrifugation.
  • Panel A shows the protein concentrations measured according to the heating temperature.
  • Panel B shows a SDS-PAGE result for samples loaded at respective heating temperatures at the same volume.
  • Panel C is a Western blotting result to analyze L1 proteins for samples loaded at respective heating temperatures at the same volume.
  • Panel D is a Western blotting result to analyze L1 proteins for samples quantified at respective heating temperatures when the samples was loaded at the same protein amount. Accordingly, panel D shows a purity of the L1 proteins. According to the result, it was confirmed that HPV16 L1 proteins remained in the cell homogenate at a heating temperature of 60° C., and the purity of the L1 protein was increased when the cell homogenate was heated at 35 to 50° C.
  • FIG. 26 shows an effect as a function of heating temperature in the step of heating/chilling after treatment with a reducing agent in the purification of HPV18 L1 proteins according to T-5 method. Detail descriptions of each panel are the same as for FIG. 25 . According to the result, it was confirmed that HPV18 L1 proteins remained in the cell homogenate up to a heating temperature of 65° C., and a purity of the L1 proteins was increased when the cell homogenate was heated at 45 to 55° C.
  • FIG. 27 shows the effect of the chilling step in heating/chilling after treatment with a reducing agent in the purification of HPV16 L1 VLP according to the T-5 method.
  • Panel A shows a difference in protein concentration between a sample which was heated at 45 to 50° C. and chilled (heating & chilling) and a sample which was not chilled (heating).
  • Panel B shows amounts of L1 proteins analyzed by Western blotting for a sample heated at 45° C. and chilled and a sample which was not chilled.
  • Panel C shows amounts of L1 proteins analyzed by Western blotting for a sample heated at 50° C. and chilled and a sample which was not chilled. This result shows that contaminating proteins were removed by precipitation through the chilling step, and in this operation, loss of the L1 proteins did not occur.
  • FIG. 28 shows the effect of the chilling step in heating and chilling after treatment with reducing agent in the purification of HPV16 L1 VLP according to the T-5 method. It shows a difference between a sample which was heated at 45° C. and chilled (HC) and a sample which was not chilled (H), which was analyzed by SDS-PAGE and Western blotting. According to the results of the SDS-PAGE and Western blotting, it was confirmed that the L1 proteins were not reduced after the chilling step while the contaminating proteins were reduced.
  • FIG. 29 shows numerical values of band intensities of contaminating proteins protein 1, protein 2 and protein 3 detected by SDS-PAGE of FIG. 28 . This shows that the concentrations of the contaminating proteins were reduced by the chilling step.
  • FIG. 30 shows results obtained by purifying HPV16 L1 VLPs and HPV18 L1 VLPs at a heating temperature of 60° C. according to a T-5 method.
  • Panel A shows an SDS-PAGE result for first chromatography.
  • LS indicates a sample loaded onto a column, and FT indicates a sample flowing through without being bound to a column. Elution indicates a fraction eluted after binding to a column resin.
  • Arrows indicate locations of HPV16 L1 and HPV18 L1.
  • Panel B shows SDS-PAGE results for HPV16 L1 and HPV18 L1 finally purified after performing the first and second chromatography.
  • Panel C is a transmission electron microscopy for a product finally purified. According to the electron microscopy result, it was confirmed that purified L1 proteins form VLPs.
  • FIG. 31 shows a result obtained by purifying HPV18 L1 by first chromatography according to a T-5 method. Each fraction was analyzed by SDS-PAGE. LS indicates a loading sample, FT indicates a flow-through, W indicates a fraction while a column is being washed, and E indicates a fraction of protein, which was attached to a column, eluted by addition of a buffer solution including 1M NaCl. It was shown that HPV18 L1 was purified with high purity by chromatography after treatment with a reducing agent and heating/chilling.
  • FIG. 32 shows a SDS-PAGE result for a HPV18 L1 protein fraction eluted after second cation-exchange chromatography that was performed using elution fraction of first chromatography of FIG. 31 .
  • LS, FT and W are the same as described above. Proteins attached to a column resin were sequentially eluted with a buffer solution containing 0.6, 0.7, 0.8, 0.9 or 1 M NaCl. It was confirmed that the HPV18 L1 proteins were eluted in 0.9 M and 1 M NaCl fractions.
  • FIG. 33 shows a DLS result for HPV18 L1 VLPs purified by a T-5 method.
  • the DLS of the T-5 HPV18 L1 VLPs was analyzed using an ELS-Z2 system.
  • FIG. 34 shows results obtained by purifying HPV58 L1 by a T-5 purification method.
  • Panel A shows an SDS-PAGE result for an L1 protein fraction eluted by first chromatography.
  • Panel B shows an SDS-PAGE result for second chromatography. Detailed description of Panel B is the same as described in FIG. 32 .
  • FIG. 35 shows SDS-PAGE and Western blotting results for HPV58 L1 proteins finally recovered by first and second chromatographies according to a T-5 purification method. It shows that HPV58 L1 can be successfully purified by a method for treating with reducing agent followed by performing heating/chilling steps.
  • FIG. 36 shows a DNA sequence coding for an HPV16 L1 protein (HPV16 L1 NG2). Saccharomyces cerevisiae was transformed with expression vector harboring the HPV16 L1 NG2 gene. The transformed cells were used to express and purify the HPV16 L1 protein. A nucleic acid sequence of HPV16 L1 was noticed as accession no. KC792555.1 of GenBank.
  • FIG. 37 shows a DNA sequence coding for an HPV18 L1 protein (HPV18 L1 NG3). Saccharomyces cerevisiae was transformed with expression vector harboring the HPV18 L1 NG3 gene. The transformed cells were used to express and purify the HPV18 L1 protein. A nucleic acid sequence of HPV18 L1 was noticed as accession no. KC792556.1 of GenBank.
  • FIG. 38 shows amino acid sequences of HPV16 L1 and HPV18 L1 encoded by the HPV16 L1 NG2 and HPV18 L1 NG3, respectively.
  • Saccharomyces cerevisiae S. cerevisiae Y2805 expressing an HPV L1 protein was cultured according to a conventionally known method [25].
  • HPV L1 protein-expressing cells were plated on a uracil-free synthetic complete medium, that is “SD-ura”, and cultured for four or five days. A single colony inoculated an SD-ura liquid medium and was cultured for two days.
  • the cultured transformed cells were cultured in an YPDG medium containing 1% yeast extract (Duchefa, Netherlands), 2% peptone (Duchefa), 7% glucose (Duchefa) and 1% galactose (Duchefa).
  • the cultured cells were centrifuged to remove the medium, and washed with phosphate-buffered saline (PBS). The washed cells were harvested by centrifugation again, and stored at ⁇ 70° C. before the purification of the protein.
  • PBS phosphate-buffered saline
  • HPV VLPs Purification of HPV VLPs according to a T-1 method was performed by heparin chromatography after proteins in a homogenate were recovered by ammonium sulfate precipitation as a pellet according to a conventionally-known method [22,23,24].
  • a HiTrapTM Heparin HP GE Healthcare, USA
  • a sample recovered from the steps for removing ammonium sulfate and removing precipitated contaminants was dialyzed against PBST containing 0.325 M NaCl, and passed through a heparin resin equilibrated with PBST containing 0.325 M NaCl.
  • the heparin resin was washed with five resin-bed volumes of the buffer (PBS containing 0.325 M NaCl), and the proteins bound with the resin were eluted by linear gradient to increase the concentration of NaCl from 0.325 to 2 M.
  • PBS containing 0.325 M NaCl
  • HPV L1 those including HPV L1 were selected by SDS-PAGE.
  • the purified HPV L1 VLPs were dialyzed against PBS containing 0.01% Tween 80 and 0.33 M NaCl (PBST).
  • HPV L1 protein-expressing cells were mixed in a disruption buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2). The cell mixture was mixed again with 0 5 mm glass beads (Biospec Product, USA), and the cells were disrupted by vortexing. Cell debris was removed by centrifugation at 12000 ⁇ g for 15 minutes. Before column chromatography, cell lysates were prepared by two different methods. The first method includes adding ⁇ -ME to the cell lysate to have a final concentration of ⁇ -mercaptoethanol ( ⁇ -ME, Sigma, USA) of 4 to 6 wt %, and adjusting pH to 7.0 to 7.3.
  • ⁇ -ME ⁇ -mercaptoethanol
  • the second method includes dialyzing the cell homogenate against the cell disruption buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 to 6 hours, and adding ⁇ -ME to the cell lysate to have a final concentration of ⁇ -ME ranging 4-6%. Subsequently, the ⁇ -ME-added cell lysate was stayed in a constant temperature water bath at 25 to 65° C. for 30 to 50 minutes, and chilled on ice for 30 minutes to 3 hours or at 4° C. for 16 hours. The chilled cell lysate was centrifuged at 12000 ⁇ g for 15 minutes to remove precipitated contaminants.
  • the cell disruption buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2
  • a cell lysate prepared by heating and chilling after treatment with reducing agent was passed through a heparin resin (HiTrapTM Heparin HP, GE Healthcare, USA or POROS® 50 HE, Applied Biosystems, USA) or a cation-exchange resin (POROS® XS, Applied Biosystems, USA). Before the cell homogenate was passed through the heparin resin or cation-exchange resin, the resin was equilibrated with a disruption buffer solution (10 mM sodium phosphate dibasic, 0.15 to 0.48 M NaCl, 1.7 mM EDTA, 0.01% Tween 80, pH 7.2) containing 4 to 6% ⁇ -ME.
  • a disruption buffer solution (10 mM sodium phosphate dibasic, 0.15 to 0.48 M NaCl, 1.7 mM EDTA, 0.01% Tween 80, pH 7.2
  • the cell homogenate was loaded onto a resin, and heparin or cation exchange resin was washed with five or more than five resin-bed volumes of wash buffer (PBST containing 0.35 to 0.48 M NaCl and 5% ⁇ -ME).
  • the HPV L1 proteins bound with heparin resin were eluted with PBST containing 1M NaCl and 5% ⁇ -ME, or by successive addition of buffer solutions prepared to have final NaCl concentrations of 0.6 M, 0.7 M, 0.8 M, 0.9 M and 1 M in PBST containing 5% ⁇ -ME.
  • the elution solution including L1 proteins was concentrated using Amicon Ultra (Millipore, USA), and dialyzed against PBST containing 1 M NaCl and 0.2 M ammonium sulfate for 20 to 24 hours.
  • the solution dialyzed after the first chromatography was additionally dialyzed against PBST containing 0.3 to 0.42 M NaCl, passed through a heparin resin or a cation-exchange resin to perform the second chromatography.
  • a heparin resin or a cation-exchange resin used for the second chromatography were the same as those used in the first chromatography.
  • the heparin/cation exchange resin was equilibrated with PBST containing 0.42 M NaCl prior to sample loading. After the sample loading, the resin was washed with five or more than five resin-bed volumes of PBST containing 0.42 M NaCl.
  • HPV L1 proteins bound with the heparin/cation exchange resin were eluted by increase of concentration of NaCl from 0.42 to 2.0 M, or by successive addition of buffer solutions prepared to have concentrations of NaCl of 0.6 M, 0.7 M, 0.8 M, 0.9 M and 1 M.
  • An elution pattern for the chromatography was monitored at a wavelength of 280 nm, and collected using an Autochro-2000 program (Young Lin Instrument Co., South Korea).
  • An L1 protein-eluted fraction was collected, concentrated using Amicon Ultra (Millipore, USA), and dialyzed against PBST containing 0.33 M NaCl.
  • cells were prepared by disruption as described above.
  • the prepared homogenate was dialyzed against a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 to 6 hours.
  • Precipitated contaminants of the dialyzed sample were removed by centrifugation at 12000 ⁇ g for 10 minutes, and then the preparation passed through a heparin resin equilibrated with the buffer solution above.
  • the heparin resin was washed with five resin-bed volumes of PBST containing 0.42 M NaCl, and proteins bound with the heparin resin were eluted with PBST containing 1 M NaCl after the wash step.
  • a purification method by treating with ⁇ -me is a purification method excluding heating & chilling step from the T-5 purification method.
  • cells expressing HPV L1 were prepared as described above, and dialyzed against a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 to 6 hours.
  • ⁇ -me was added to the dialyzed lysate to have a final concentration of 4 to 6% of ⁇ -me.
  • the heparin resin was equilibrated with a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) containing 4 to 6% ⁇ -me, and the prepared homogenate passed through the resin.
  • the heparin resin through which the lysate passed was washed with five resin-bed volumes of PBST containing 4 to 6% ⁇ -me and 0.42 M NaCl. Proteins attached to the heparin resin were eluted with PBST containing 4 to 6% ⁇ -me and 1 M NaCl.
  • a heating & chilling method is a purification method excluding the ⁇ -me treatment step from a T-5 purification method.
  • HPV L1 proteins For purification of HPV L1 proteins, cells were disrupted and dialyzed against a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 to 6 hours. The dialyzed lysate was treated at 37 to 45° C. for 30 minutes, and chilled on ice for 30 minutes to 3 hours.
  • Precipitated contaminants of the lysate subjected to heating/chilling were removed by centrifugation at 12000 ⁇ g for 10 minutes, and passed through a heparin resin equilibrated with a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2). Afterward, the heparin resin was washed with 5 resin volumes of PBST containing 0.42 M NaCl, and proteins bound with the resin after washing step were eluted with PBST containing 1 M NaCl.
  • a buffer solution 10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2
  • HPV L1 expression yeasts were prepared by disruption as described above, and proteins were precipitated by saturating 45% ammonium sulfate. The precipitated proteins were resuspended using PBST, and dialyzed against a buffer solution (10 mM sodium phosphate dibasic, 0.65M NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 hours. Purification of the dialyzed fraction was performed by SEC as first chromatography.
  • the prepared sample passed through a superose-6 resin (1.5 ⁇ 32 cm, GE Healthcare, USA) equilibrated with PBST containing 0.65 M NaCl (first chromatography) [36].
  • An elution pattern of the SEC was detected with a wavelength of 280 nm, and collected using an Autochro-2000 program (Young Lin Instrument Co., South Korea).
  • Purification using ammonium sulfate precipitation is a method in which heating & chilling step after ⁇ -me treatment in T-5 method is replaced with ammonium sulfate precipitation.
  • ammonium sulfate precipitation and removal of precipitated contaminants were performed according to a conventionally known method [22, 24]. Proteins in a cell lysate were saturated with 45% ammonium sulfate to precipitate, and contaminating substances were removed (removal of precipitated contaminants).
  • a sample prepared above before chromatography was dialyzed against a buffer solution (10 mM sodium phosphate dibasic, 150 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80 pH 7.2) for 4 hours.
  • a heparin resin was equilibrated with the same buffer solution as used above, and then the dialyzed sample passed through the resin (first chromatography). Afterward, the resin was washed with five resin-bed volumes of PBST containing 0.42 M NaCl, and binding proteins were eluted with PBST containing 1 M NaCl. For second chromatography, an elution fraction was dialyzed against a buffer solution containing 0.33 M NaCl. Afterward, the second chromatography of the SEC was performed in the same manner as described above.
  • a concentration of proteins was measured with bovine serum albumin (BSA; Pierce, USA) as a standard by a protein quantification kit (Bio-Rad Laboratories, USA).
  • BSA bovine serum albumin
  • SDS-PAGE was performed according to a Laemmli's method [35], and Western blotting was performed by a known method [26].
  • a protein developed on an SDS-PAGE gel was visualized by staining.
  • An HPV L1 protein was detected using a rabbit anti-HPV 16 L1 serum as a primary antibody and a goat HRP-conjugated anti-rabbit IgG polyclonal antibody (HRP-conjugated goat anti-rabbit IgG, Bethyl, USA) as a secondary antibody [26].
  • a band intensity was measured by National Institute Health (NIH) open source software Image J, and estimated according to a known method [25].
  • NASH National Institute Health
  • Purified HPV 16 L1 proteins were adsorbed onto a carbon-coated grid, and stained with phosphotungstic acid or uranyl acetate.
  • a transmission electron microscope image was taken using TEM200CX (JEOL, Japan) at a final magnification of 150,000 ⁇ .
  • DLS for HPV VLPs was performed by a known method [26]. Purified HPV VLPs were diluted in PBST containing 0.13 M NaCl to have a concentration of 0.13 mg/ml, and analyzed using a DLS-700 system (Otsuka Electronics, Japan) or an ELS-Z2 system (Otsuka Electronics, Japan).
  • a reactivity of a monoclonal antibody to HPV VLPs was analyzed according to a conventionally known method [26].
  • a 96-well enzyme-linked immunosorbent assay (ELISA) plate was coated with 400 ng of purified HPV VLPs.
  • the HPV L1 VLPs purified by different methods were used as coatings after SDS-PAGE was performed to confirm that an amount of L1 proteins of HPV L1 VLPs purified in each method was quantitatively the same.
  • the VLP-coated plate was blocked with PBS-T 20 containing 3% bovine serum albumin (PBS containing 0.05% Tween 20) at room temperature for 2 hours.
  • the resulting antibodies were washed with PBS-T three times, a HRP-conjugated anti-mouse IgG antibody (Bethyl, USA) was diluted in PBS-T 20 containing 0.3% bovine serum albumin at a ratio of 1:5000, and reacted on a plate at 37° C. for 40 minutes.
  • the plate was washed with PBS-T 20 five times, and a color reaction was performed.
  • the color reaction was performed using o-phenylenediamine (Sigma, USA), and an optical density was detected at 492 nm.
  • HPV16 L1 To evaluate immunogenicity of HPV16 L1 , a 6-week old Balb/c mouse was used (Orientbio. South Korea). To immunize mice with HPV L1 proteins, purity and concentration of a L1 protein were confirmed according to the known protein quantification method and SDS-PAGE method. The mouse was immunized by subcutaneous injection four times at two week intervals. For a single immunization, 1 ng of L1 protein in combination with 200 ⁇ g aluminum hydroxide (Sigma, USA) was injected subcutaneously. 1 ng of a protein was based on a quantification result for T-5 HPV16 L1 VLPs. HPV16 L1 protein purified by another method was quantified using T-5 HP16 L1 as a standard substance.
  • FIG. 1 A T-5 purification process is shown in FIG. 1 .
  • Loading samples for first heparin chromatography were prepared into two types including the case in which dialysis was performed and the case in which dialysis was not performed.
  • dialysis was performed in a cell disruption buffer solution, and then a reducing agent ( ⁇ -ME) was added, and when dialysis was not performed, a reducing agent was directly added to the homogenate.
  • two samples were heated at 37 to 42° C., and left on ice for 30 to 50 minutes to chill down to approximately 0° C. A contaminating substances precipitated resulting from the heating/chilling step were removed, and heparin chromatography was performed.
  • FIGS. 2 and 3 show heparin chromatography results for the dialyzed sample and the non-dialyzed sample, which were analyzed by SDS-PAGE. It appeared that L1 proteins were obtained with high purity in both cases.
  • FIG. 4 a shows a purified result obtained by the second heparin chromatography.
  • the L1 proteins bound with a heparin resin were eluted by linearly increasing of a NaCl gradient ( FIG. 4 b ).
  • As the result of SDS-PAGE, L1 proteins flowing through without being bound to the heparin resin were not observed in an FT fraction. It was confirmed that the L1 proteins bound with the heparin resin were eluted by an increase in the linear gradient (Fractions 11-17).
  • FIG. 4 a shows a purified result obtained by the second heparin chromatography.
  • the L1 proteins bound with a heparin resin were eluted by linearly increasing of a NaCl gradient ( FIG. 4 b ).
  • As the result of SDS-PAGE, L1 proteins flowing through without being bound to the heparin resin were not observed in an FT fraction. It was confirmed that the L1 proteins bound with the heparin resin were elute
  • 4 b shows eluted substances detected at a wavelength of 280 nm in the heparin chromatography.
  • the eluted proteins were not observed in FT (flow-through), but, when detected at 280 nm, it was confirmed that a considerable amount of substances was flowed through in the FT. Accordingly, it was confirmed that contaminating substances, other than the L1 proteins, were removed by the second heparin chromatography.
  • T-5 HPV16 L1 VLP A purity of L1 protein collected by the second heparin chromatography (T-5 HPV16 L1 VLP) was compared with a purity of L1 proteins purified by the conventionally known method [22.24] (T-1 HPV16 L1 VLP).
  • the T-1 purification method is shown in FIG. 1
  • an SDS-PAGE result for heparin chromatography of the T-1 purification method is shown in FIG. 5 .
  • LS indicates a sample loaded onto a column (loading sample).
  • FIG. 6 a shows result comparing purities of HPV16 L1 VLP purified by the T-1 and T5 methods.
  • T-1 HPV16 L1 VLP and T-5 HPV16 L1 VLP were loaded at 500 ng, 250 ng, 125 ng, and 62 ng per well after the quantification of the proteins, and after fractionation of that from SDS-PAGE, the result thereof was visualized by staining. From two VLPs, a high purities of a 55 kDa L1 bands were observed. However, it was seen that an intensity of the L1 band of T-5 HPV 16 VLP was higher than that of T-1 HPV 16 VLP.
  • FIG. 6 b shows values of two experiments conducted in FIG. 6 a , which were represented as mean ⁇ standard deviation.
  • an intensity of L1 band of T-5 HPV 16 VLP loaded at 500 ng was set to 100%. According to the results, it was confirmed that the purity of T-5 HPV16 L1 VLPs was higher than that of T-1 HPV16 L1 VLPs.
  • FIG. 7 shows an SDS-PAGE result of L1 amounts of two types of VLPs, which were adjusted to be the same.
  • FIG. 8 shows results of electron microscopy for T-1 HPV 16 VLPs and T-5 HPV 16 VLPs. It was confirmed that a size of the T-5 HPV16 L1 VLP ranges from 40 to 65 nm, and a size of the T-1 HPV16 L1 VLP ranges from 20 to 50 nm. Accordingly, a type of the HPV16 L1 VLP purified by the T-5 method had characteristics different from those of a type of the HPV16 L1 VLP purified by the T-1 method. It was known that a size of an HPV virion naturally occurring was 50 to 60 nm [29,30]. Such a result denotes that the size of the T-5 HPV 16 VLP is closer to an original HPV size.
  • FIG. 9 shows a representative result of analyzing purified T-1 HPV16 L1 VLPs and T-5 HPV 16 L1 VLPs using a DLS-700 system.
  • FIG. 10 shows a representative result of analyzing HPV16 L1 VLPs using an ELS-Z2 system.
  • VLP size was represented as mean ⁇ standard deviation.
  • the T-1 HPV16 L1 VLPs were distributed between 29 and 438 nm, and T-5 HPV16 L1 VLPs were distributed between 17 to 233 nm. Accordingly, hydrostatic distributions according to sizes of two types of VLPs were different from each other.
  • FIG. 9 shows a representative result of analyzing purified T-1 HPV16 L1 VLPs and T-5 HPV 16 L1 VLPs using a DLS-700 system.
  • FIG. 10 shows a representative result of analyzing HPV16 L1 VLPs using an ELS-Z2 system.
  • VLP size was represented as mean ⁇ standard deviation.
  • FIG. 10A shows intensity profiles of DLS of the two VLPs.
  • FIG. 10B shows polydispersity indexes (P.I.) of the two types of VLPs.
  • P.I. polydispersity indexes
  • Reactivity of an anti-HPV 16 L1 monoclonal antibody to HPV 16 VLP is used as an important criterion to evaluate structural superiority of HPV16 L1 VLPs and ability of those to induce the neutralizing antibodies [26, 32-34].
  • Reactivity of T-1 HPV16 L1 VLPs and T-5 HPV16 L1 VLPs against the monoclonal antibodies were compared using antibodies H16.V5 and H16.E70 which the most generally used to evaluate these properties [26].
  • An increase in reactivity to the two antibodies is closely related with an increase in immunogenicity [26, 36].
  • FIG. 11 it was confirmed that the reactivity of T-5 HPV16 VLPs against two types of antibodies are significantly higher than those of T-1 HPV16 VLPs.
  • T-1 HPV16 L1 VLP a purity of L1 proteins of T-1 HPV16 L1 VLP is lower than that of T-5 HPV16 L1 VLP ( FIG. 6 a , FIG. 6 b ).
  • the amount of T-1 HPV16 L1 VLP was adjusted to that of T-5 HPV16 L1 VLPs, and the amounts of L1 proteins were evaluated by SDS-PAGE ( FIG. 7 ).
  • 1 ng of HPV16 L1 VLP was injected in combination with aluminum hydroxide.
  • the 1 ng of protein amount (the value obtained from Bradford protein assay) quantified from T-5 HPV16 L1 VLPs was set as a standard.
  • the HPV16 L1 VLPs purified by the T-1 or T-5 purification methods was subcutaneously injected into mice four times at two weeks intervals. Titers of anti-HPV16 L1 IgG in sera detected after third and fourth immunizations are shown in FIG. 12 . After the third immunization, the T-5 HPV16 L1 VLP-immunized group had a median value of 450, whereas the T-1 HPV16 L1 VLP-immunized group had a median value of 0. After the fourth immunization, the T-5 HPV16 L1 VLP-immunized group had a median value of 4050, whereas the T-1 HPV16 L1 VLP-immunized group had a median value of 300. Accordingly, it was confirmed that the T-5 HPV16 L1 VLPs induced 10 times or more than 10 times higher level of anti-HPV16 L1 IgG antibody titer than T-1 HPV16 L1 VLPs did.
  • FIG. 13 After the fourth immunization, an anti-HPV16 neutralizing antibody activities in a mouse sera were detected ( FIG. 13 ).
  • a T-5 HPV16 L1 VLP-immunized group had a neutralizing activity (median value) of 78% while a T-1 HPV16 L1 VLP-immunized group had a neutralizing activity of 33%. Between the two groups, the neutralizing activity showed a significant difference.
  • Tables 1 and 2 show a protein amount and a rate of removing contaminating proteins of a loading sample in each condition for the heparin chromatography in the first and second experiments, respectively.
  • Table 1 shows a protein amount and a rate of removing contaminating proteins of a loading sample in each condition for the heparin chromatography in the first and second experiments, respectively.
  • Table 2 shows a protein amount and a rate of removing contaminating proteins of a loading sample in each condition for the heparin chromatography in the first and second experiments, respectively.
  • Table 1 show a protein amount and a rate of removing contaminating proteins of a loading sample in each condition for the heparin chromatography in the first and second experiments, respectively.
  • Table 1 shows a protein amount and a rate of removing contaminating proteins of a loading sample in each condition for the heparin chromatography in the first and second experiments, respectively.
  • Table 1 shows a protein amount and a rate of removing contaminating proteins of a
  • FIG. 14 shows SDS-PAGE and Western blots of fractions obtained after the first heparin chromatography when loaded at the same volumes (5 ⁇ L, 2.5 ⁇ L, 1.2 ⁇ L). It was confirmed that in the non-treatment, the amount of obtained L1 proteins was considerably decreased. It was also confirmed that since many foreign proteins were included in the recovered solution, purity was also very low. It was confirmed that, when only a reducing agent, that is, ⁇ -ME, was treated, the amount of recovered L1 proteins was increased, but due to a large amount of contaminating proteins in the harvested solution, a purity was not high, either.
  • a reducing agent that is, ⁇ -ME
  • the L1 protein was identified as a major band in SDS-PAGE and Western blotting, and thus a recovery rate, as well as the purity, of the L1 proteins was also high.
  • FIG. 16 shows SDS-PAGE results of elution fractions obtained from the first heparin chromatography in a non-treatment, when only heating/chilling was performed (heating & chilling method), and when heating/chilling was performed after treatment with a reducing agent (T-5, ⁇ -ME+ heating & chilling).
  • Fractions collected after the first heparin chromatography were loaded at the same volumes (5 ⁇ L, 2.5 ⁇ L, 1.2 ⁇ L), and analyzed by SDS-PAGE and Western blotting.
  • amounts of recovered L1 proteins were considerably decreased.
  • due to large amounts of contaminating proteins in the collected solution the purity was also very low.
  • the L1 protein was confirmed as a major band in SDS-PAGE and Western blot, and thus a recovery rate, as well as the purity, of the L1 proteins was also high.
  • a purification process using ammonium sulfate precipitation is shown in FIG. 17 .
  • a purification method by ammonium sulfate precipitation is designed by replacing the heating & chilling step after ⁇ -ME treatment in the T-5 purification method with ammonium sulfate precipitation, and is distinguished from the T-1 purification method shown in FIG. 1 .
  • the method by ⁇ -ME treatment ⁇ -ME method) is the same as described in the above example. Proteins eluted from first chromatography were loaded at the same volumes (10, 5, 2.5 ⁇ l and analyzed by SDS-PAGE and Western blotting ( FIG. 18 and FIG. 19 ).
  • FIG. 17 A size-exclusion chromatography (SEC) process is shown in FIG. 17 .
  • the SEC method was based on a conventionally known method [20].
  • the ammonium sulfate precipitation was the same as described above.
  • a representative SEC result is shown in FIG. 20 .
  • HPV16 L1 proteins after SEC were eluted with high purity in fractions 3 to 9, and the fractions were used to compare the purity of L1 proteins by first chromatography ( FIG. 21 ). Purities of L1 proteins in first chromatography elution fractions by SEC, ammonium sulfate precipitation, and heating/chilling after ⁇ -ME treatment ⁇ -ME+ heating & chilling) were compared ( FIG. 21 ).
  • FIG. 22 shows SDS-PAGE and Western blotting results for the elution fractions of second chromatography which were performed using elution fraction of first chromatography presented in FIG. 21 .
  • Elution fraction of each condition in second chromatography was loaded at the same volume (2, 1, or 0.5 ⁇ L). According to the analysis of the elution fractions of second chromatography, it was confirmed that when heating/chilling was performed after ⁇ -ME treatment, the yield of the L1 proteins was the highest.
  • VLPs yielded by the method conducted by heating/chilling after ⁇ -ME treatment has a higher purity than those purified by a conventionally known method such as SEC and ammonium sulfate precipitation, and that has an excellent antigenic characteristic.
  • VLPs 1 ng each of the three types of HPV16 L1 VLPs finally purified in FIG. 23 was used for immunization. Before immunization, as shown in FIG. 23A , VLPs was adjusted to have the same concentrations. In the immunization of mice, 1 ng of VLP was injected in combination with 200 ⁇ g of aluminum hydroxide Immunization was performed four times at two week intervals.
  • FIGS. 25 and 26 show an effect of removing contaminating proteins according to a heating temperature after treatment with a reducing agent in the purification of HPV16 L1 VLPs and HPV18 L1 VLPs.
  • the homogenate was treated with ⁇ -ME, and reacted at room temperature (starting), 25, 30, 35, 40, 45, 50, 55, 60, or 65° C. for 15 minutes. Afterward, the sample was left on ice for 1 hour, and centrifuged at 12000 ⁇ g to remove precipitated contaminants. A concentration of proteins in the sample resulting from which precipitated contaminants were removed was measured and shown in Panel A. Each sample was loaded at the same volume, and the results analyzed by SDS-PAGE were presented in Panel B.
  • FIGS. 25A , 25 B, 26 A, and 26 B It is apparent that a removing rate of contaminating proteins according to heating was highly increased at 35° C. or more ( FIGS. 25A , 25 B, 26 A, and 26 B).
  • FIGS. 25C and 26C it was confirmed that HPV16 L1 proteins remained in the homogenate at a heating temperature of 65° C. and 60° C., and HPV18 L1 proteins remained in the homogenate at a heating temperature of 60° C.
  • the purity of the HPV16 L1proteins was high at temperature ranging 35 to 50° C. ( FIG. 25D ), and the purity of the HPV18 L1 proteins was the highest at temperature ranging 45to 60° C. ( FIG. 26D ).
  • the homogenate was heated at 35 to 60° C.
  • contaminating proteins were removed, while the L1 proteins maintained the stability.
  • the thermal stability of the L1 protein can facilitate separation of the L1 proteins from the contaminating proteins by the heating step under the condition of treating with reducing agents.
  • FIG. 27 shows comparison results between the condition in which only heating was performed after treatment with a reducing agent (heating) and the condition in which chilling was performed after heating (heating & chilling).
  • Panel A shows concentrations of proteins measured when only heating was performed, and when chilling was performed after heating.
  • Panels B and C showed Western blotting results obtained when only heating was performed at 45° C. and 50° C., and when chilling was performed after heating (heating and chilling).
  • For Western blotting a sample for each condition was loaded at the same volumes (5, 2.5 and 1.25 ⁇ l). As shown in Panel A, it was confirmed that a total amount of proteins was reduced when chilling was performed after heating, compared to when only heating was performed. Meanwhile, it was confirmed that the amount of L1 proteins was not decreased ( FIGS. 27B and 27C ). Such results show that the purity of L1 proteins was increased by the chilling.
  • FIG. 28 shows SDS-PAGE and Western blotting results when only heating was performed after treatment with a reducing agent (H) and when chilling was performed after heating (HC).
  • H reducing agent
  • HC chilling was performed after heating
  • Homogenates of yeast cells expressing HPV16 L1 and HPV18 L1 were treated with a reducing agent, heated at 60° C., and chilled to attempt to perform VLP purification.
  • the panel A in FIG. 30 shows SDS-PAGE analysis results for first chromatography.
  • LS and FT indicate a loading sample and a flow-through, respectively. Elution indicates a fraction formed by L1 proteins eluted.
  • Panel B shows SDS-PAGE analysis results for the final product of second chromatography. Each type of L1 proteins was purified with high purity.
  • Panel C shows transmission electron microscopy results for the final purification product. It was confirmed that VLPs were formed after L1 proteins were purified after being heated at 60° C.
  • FIG. 31 shows SDS-PAGE results for first chromatography when HPV18 L1 VLPs were purified by a T-5 method.
  • Cell homogenate was treated with a reducing agent before first heparin chromatography, heated at 45° C., and chilled on ice.
  • FIG. 31 shows that contaminating proteins were effectively removed by the first heparin chromatography.
  • FIG. 32 shows SDS-PAGE results for second cation-exchange chromatography. In second chromatography, the contaminating substances were eluted from fractions including 0.6 M and 0.7 M NaCl, and L1 proteins were eluted from fractions including 0.9 M and 1 M NaCl. Accordingly, it was confirmed that HPV18 L1 proteins were purified with high purity by a T-5 method.
  • FIG. 33 shows dynamic light scattering (DLS) results for finally purified T-5 HPV18 L1 VLPs.
  • DLS dynamic light scattering
  • FIG. 34A shows SDS-PAGE results for HPV58 L1 fractions separated by a first chromatography according to the T-5 method. According to the first chromatography, a HPV58 L1 protein was detected as a major band.
  • FIG. 34B shows SDS-PAGE results for HPV58 L1 VLPs separated by second chromatography. The HPV58 L1 proteins were eluted from all of fractions including 0.6 M, 0.7 M, 0.8 M, 0.9 M, and 1 M NaCl.
  • FIG. 35 shows SDS-PAGE and Western blotting results for HPV58 L1 VLPs finally purified by the T-5 method. According to the T-5 method, it was confirmed that HPV58 L1 VLPs were purified with high purity.
  • the present invention relates to a method of purifying HPV L1 proteins with high purity and high efficiency. According to the purification method of the present invention, a purification purity of HPV L1 proteins can be considerably increased, and VLPs of the purified HPV L1 protein form a structure more similar to that of a native HPV virion, and thus have very excellent immunogenicity.
  • Kim H J, Lim S J, Kim J Y, Kim S Y, Kim H-J (2009) A method for removing contaminating protein during purification of human papillomavirus type 18 L1protein from Saccharomyces cerevisiae . Arch Pharm Res 32: 1759-1766.
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